The objective of this research proposal is to develop and characterize novel synthetic ligands for thyroid hormone receptors (TRs). TRs belong to the nuclear receptor superfamily of ligand-activated transcription regulators and the physiological effects of thyroid hormone in different tissues are a result of TR-mediated regulation of thyroid hormone responsive genes. There are two genes encoding different TRs, TRalpha and TRbeta, and these two TRs are expressed at different levels in different tissues. We have developed a novel synthetic thyroid hormone analog called GC-1 which is selective for TRbeta. We have characterized the binding and receptor-activation properties of GC-1 and demonstrated that the TRbeta selectivity is high enough to stimulate tissue-selective hormone effects in vivo. The proposed research in this renewal application builds on our success with this family of ligands and is composed of five specific aims. Aim 1 is focuses on the development of stable cell lines that express either TRalpha or TRbeta and contain a chromosomal copy of a TRE-driven reporter construct. These cell lines will facilitate screening of test ligands in a more efficient and reliable manner than the current assay that relies on transient transfection. Aims 2 and 3 deal with the development of antagonist ligands for the TR that will block activation of TR responsive genes. For these studies the GC-1 core structure will modified in different ways such that the modification will perturb an important activation surface of the liganded TR complex. These proposed modifications are based on analysis of the crystal structure of TR bound to various ligands including GC-1 and thyroid hormone. Aim 4 is centered on the development of TRalpha selective ligands. Modifications to the GC-1 core structure are proposed that are designed to form specific H-bonding interactions with TRalpha but not with TRbeta. In Aim 5 collaborative in vivo experiments are proposed to assess the activity of the synthetic test ligands in a physiologically relevant context. If successful, these studies will provide new selective agonist and antagonist ligands for TRalpha and TRbeta which will be useful as experimental probes of thyroid hormone action in different tissues. Moreover, these selective ligands could be useful as therapeutics for the treatment of human disease associated with thyroid hormone regulation.